Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naive subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and Fc gamma RI (CD64) receptor (P <= 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells (P=0.0356 and P=0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-alpha, IL-12p40 and IFN-gamma (P <= 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection. (c) 2009 Elsevier Ltd. All rights reserved.

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alpha beta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes, Here we describe a DNA plasmid encoding a polyepitope or polytope protein, which contained multiple contiguous minimal murine CTL epitopes, Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models, CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help, The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.

Reticular angiomatoid ""malignant"" fibrous histiocytoma-a case report with cytogenetics and molecular genetic analyses

Angiomatoid ""malignant"" fibrous histiocytoma is a rare sarcoma of low malignant potential that occurs most commonly in the extremities of children and young adults. Herein, we present a case of angiomatoid malignant fibrous histiocytoma with unusual histologic features arising in the mediastinum of an 80-year-old man. The tumor exhibited a reticular growth pattern and myxoid stroma. The tumor cells expressed epithelial membrane antigen and desmin. Cytogenetic analysis revealed the translocation t(2;22)(q33;q12). Molecular genetic analysis confirmed the rearrangement of the EWSR1 locus and the presence of the EWSR1/CREB1 fusion. This report expands the clinicopathologic spectrum of angiomatoid malignant fibrous histiocytoma and underscores the value of integrating morphologic, immunophenotypic, and molecular findings in the identification of its unusual morphologic variants. (C) 2011 Elsevier Inc. All rights reserved.

Clear cell odontogenic carcinoma: case report with immunohistochemical findings adding support to the challenging diagnosis

Clear cell odontogenic carcinoma (CCOC) is a rare odontogenic tumor associated with aggressive clinical behavior, metastasis, and low survival. We report a case of CCOC affecting the mandible of a 39-year-old man. The tumor presented a biphasic pattern composed of clear cell nests intermingled with eosinophilic cells and separated by collagenous stroma. Immunoreactivity to cytokeratin (CK), specifically AE1/AE3 and CK 8, 14, 18, and 19 was found, as well as to epithelial membrane antigen (EMA). The tumor cells were negative for S100 protein, CK 13, vimentin, smooth muscle actin, laminin and type IV collagen. Low labeling indices for the proliferation markers Ki-67 and proliferating cell nuclear antigen and to p53 protein might predict a favorable prognosis for the lesion. A surgical resection was performed, followed by adjuvant radiotherapy. A 2-year follow-up has shown no signs of recurrence. The significance of histochemical and immunohistochemical resources in the correct diagnosis of CCOC is analyzed.

Towards a blood-stage vaccine for malaria: Are we following all the leads?

Although the malaria parasite was discovered more than 120 years ago, it is only during the past 20 years, following the cloning of malaria genes, that we have been able to think rationally about vaccine design and development. Effective vaccines for malaria could interrupt the life cycle of the parasite at different stages in the human host or in the mosquito. The purpose of this review is to outline the challenges we face in developing a vaccine that will limit growth of the parasite during the stage within red blood cells - the stage responsible for all the symptoms and pathology of malaria. More than 15 vaccine trials have either been completed or are in progress, and many more are planned. Success in current trials could lead to a vaccine capable of saving more than 2 million lives per year.

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-I and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.

Immunity to asexual blood stage malaria and vaccine approaches

The development of a malaria vaccine seems to be a definite possibility despite the fact that even individuals with a life time of endemic exposure do not develop sterile immunity. An effective malaria vaccine would be invaluable in preventing malaria-associated deaths in endemic areas, especially amongst children less than 5 years of age and pregnant women. This review discusses our current understanding of immunity against the asexual blood stage of malaria - the stage that is responsible for the symptoms of the disease - and approaches to the design of an asexual blood stage vaccine.

Detection of West Nile Virus Infection in birds in the United States by blocking ELISA and immunohistochemistry

A blocking ELISA targeting an immunodominant West Nile epitope on the West Nile Virus NS1 protein was assessed for the detection of West Nile-specific antibodies in blood samples collected from 584 sentinel chickens and 238 wild birds collected in-New Jersey from May-December 2000. Ten mallard ducks (Anas platyrhynchos) experimentally infected with West Nile virus and six uninfected controls were also tested. The ELISA proved specific in detecting WNV antibodies in 9/10 chickens and 4/4 wild birds previously confirmed as positive by Plaque Reduction Neutralization test (PRNT) at the Center for Disease Control, Division of Vector Borne Diseases, Fort Collins, CO, USA (CDC). Nine out of the ten experimentally infected mallard ducks also tested positive for WN antibodies in the blocking ELISA, while 6/6 uninfected controls did not. Additionally, 1705 wild birds, collected in New Jersey from December 2000-November 2001 and Long Island, New York between November 1999 and August 2001 were also tested for WN antibodies by the blocking ELISA. These tests identified 30 positive specimens, 12 of which had formalin-fixed tissues available to allow detection of WN specific viral antigen in various tissues by WNV-specific immunohistochemistry. Our results indicate that rapid and specific detection of antibodies to WN virus in sera from a range of avian species by blocking ELISA is an effective strategy for WN Virus surveillance in avian hosts. In combination with detection of WN-specific antigens in tissues by immunohistochemistry (IHC) the blocking ELISA will also be useful for confirming WN infection in diseased birds.

Atypical disseminated cutaneous histoplasmosis in an immunocompetent child, caused by an "aberrant" variant of Histoplasma capsulatum var. capsulatum

A case of atypical disseminated cutaneous histoplasmosis in a five-year old, otherwise healthy child, native and resident in São Paulo metropolitan area is reported. Cutaneous lesions were clinically atypical. Histologic examination disclosed a granulomatous reaction but no fungal structures could be demonstrated by specific staining nor by immunohistochemical reaction. The fungus was isolated from biopsy material on two different occasions, confirming diagnosis of an unusual fungal infection. The fungus, originally thought to be a Sepedonium sp. due to the large sized, hyaline or brownish colored tuberculated macroconidia and to lack of dimorphism (yeast form at 37 °C) produce H and M antigens, visualized by the immunodiffusion with rabbit anti-Histoplasma capsulatum hyperimmune serum. Patient’s serum sample was non reactive with H. capsulatum antigen by immunodiffusion, counterimmunoelectrophoresis and complement fixation tests, and immunoenzymatic assay failed to detect the specific circulating antigen. This serum was tested negative by double immunodiffusion when antigen obtained from one of the isolated samples was used. Both cultures were sent to Dr. Leo Kaufman, Ph.D. (Mycoses Immunodiagnostic Laboratory, CDC-Atlanta/USA), who identified them as H. capsulatum by the exoantigen and gen-probe tests. Both clinic and mycologic characteristics of the present case were atypical, suggesting the fungus isolated is an “aberrant variant” of H. capsulatum var. capsulatum, as described by SUTTON et al. in 199719. Treatment with itraconazole 100 mg/day led to cure within 90 days

Detection of Cysticercus antigens and antibodies in cerbrospinal fluid of patients with chronic meningitis

Chronic meningitism is a less frequent manifestation of neurocysticercosis caused by Taenia solium cysticerci. In the present study we used Co-agglutination (Co-A), a simple and rapid slide agglutination test to detect specific Cysticercus antigen in the 67 cerebrospinal fluid (CSF) samples from patients with chronic meningitis of unknown etiology. The results were compared with that of ELISA for detection of antibodies. Among these samples four (5.97%) were positive for Cysticercus antigen by Co-A test and six (8.95%) were positive for antibodies by ELISA. Two samples were positive by both Co-A and ELISA, two were positive only by Co-A and four were positive only by ELISA. In the present study, although Cysticercus antigen and antibodies were present in CSF samples from eight (11.94%) patients, we cannot affirm that all the cases of chronic meningitis are due to cysticercosis, but for any case of chronic meningitis of unknown origin, it would be useful to consider the possibility of cysticercal meningitis.

Cellular schwannomas of the intracranial and intraspinal compartment: morphological and immunological characteristics compared with classical benign schwannomas.

The concept of cellular schwannoma as an unusual benign tumor is well established for peripheral nerves but has never been tested in neurosurgical series. In order to test the validity of this concept in cranial nerves and spinal roots we performed an analysis of the clinical and morphological characteristics of 12 cellular and 166 classical benign schwannomas. Immunohistochemical detection of antigen expression in Schwann cells including proliferating cell nuclear antigen (PCNA) was also performed. This study shows that cellular schwannomas in neurosurgical series manifest at a lower age than the classical benign variant and occur mainly in the spinal roots. Mitotic activity and sinusoidal vessels appear more frequently in cellular schwannomas and constitute with high cellularity, the most valuable criteria separating both entities. The postoperative course in both types of tumors was free of metastases or sarcomatous changes. Immunoexpression of S-100 protein, vimentin, epithelial membrane antigen and glial fibrillary acidic protein is not statistically different between the two variants. In contrast, PCNA is more highly expressed in cellular schwannomas. These These results confirm the concept that cellular schwannomas are a clinico-pathological variant of benign schwannomas and provide significant support for the introduction of this entity in neurosurgical oncology.

Natural bradykinin antagonists

Bradykinin (BK) a nonapeptide generated in plasma during tissue injury, is involved in many physiological and pathological states. Kinin actions are mediated by specific membrane receptors and involve a complex signal transducer and also second messager mechanisms. Due to its inequivocal relevance, an intensive effort has been focused in recent years to develop selective and competitive BK antagonists. Thus, the development of a new series of peptide BK antagonists has made an important contribution to the understanding of the pharmacological, physiological and pathophysiological role of BK, and this is certain to provide a firm basis for developing new drugs to relieve pain and inflammation. However, BK antagonists derived from peptide origin reported to date have limited clinical use due to their poor oral absortion and short duration of effect. Thus, considerable effort has also been made in developing stable nonpeptide BK antagonists. Up to now, most nonpeptide compounds reported to exhibit BK antagonistic activity have been derived from plants, including many flavonoids, terpenes, and also synthetic substances with various molecular structures. Amongst them, the pregnane glycoside compounds isolated from the plant Mandevilla velutina are the most promising. These compounds are effective in antognizing BK responses in a variety of preparations, and they also exhibit potent and long-lasting analgesic and anti-inflammatory activities. The exact mechanism underlying their action however, is not yet completely understood.

Aspects of immunity for the AMA-1 family of molecules in humans and non-human primates malarias

The apical membrane antigen (AMA-1) family of malaria merozoite proteins is characterised by a high degree of inter-species conservation. Evidence that the protein (PK66/AMA-1) from the simian parasite Plasmodium knowlesi was protective in rhesus monkeys suggested that the 83kDa P. falciparum equivalent (PF83/AMA-1) should be investigated for protective effects in humans. Here we briefly review pertinent comparative data, and describe the use of an eukaryotic full length recombinant PF83/AMA-1 molecule to develop a sensitive ELISA for the determination of serological responses in endemic populations. The assay has revealed surprisingly high levels of humoral response to this quantitatively minor antigen. We also show that PK66/AMA-1 inhibitory mAb's are active against merozoites subsequent to release from schizont-infected red cells, further implicating AMA-1 molecules in red cell invasion.

Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory.

Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates its genome in a membrane-associated replication complex. Nonstructural protein 4B (NS4B) induces the specific membrane alteration, designated as membranous web (MW), that harbours this complex. HCV NS4B is an integral membrane protein predicted to comprise four transmembrane segments in its central part. The N-terminal part comprises two amphipathic alpha-helices of which the second has the potential to traverse the membrane bilayer, likely upon oligomerisation. The C-terminal part comprises a predicted highly conserved alpha-helix, a membrane-associated amphipathic alpha-helix and two reported palmitoylation sites. NS4B interacts with other viral nonstructural proteins and has been reported to bind viral RNA. In addition, it was found to harbour an NTPase activity. Finally, NS4B has recently been found to have a role in viral assembly. Much work needs to be done with respect to further dissecting these multiple functions as well as providing a refined membrane topology and complete structure of NS4B. Progress in this direction should yield important insights into the functional architecture of the HCV replication complex and may reveal new opportunities for antiviral intervention against a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide.

Molecular markers and genetic diversity of Plasmodium vivax

Enhanced understanding of the transmission dynamics and population genetics for Plasmodium vivax is crucial in predicting the emergence and spread of novel parasite phenotypes with major public health implications, such as new relapsing patterns, drug resistance and increased virulence. Suitable molecular markers are required for these population genetic studies. Here, we focus on two groups of molecular markers that are commonly used to analyse natural populations of P. vivax. We use markers under selective pressure, for instance, antigen-coding polymorphic genes, and markers that are not under strong natural selection, such as most minisatellite and microsatellite loci. First, we review data obtained using genes encoding for P. vivax antigens: circumsporozoite protein, merozoite surface proteins 1 and 3α, apical membrane antigen 1 and Duffy binding antigen. We next address neutral or nearly neutral molecular markers, especially microsatellite loci, providing a complete list of markers that have already been used in P. vivax populations studies. We also analyse the microsatellite loci identified in the P. vivax genome project. Finally, we discuss some practical uses for P. vivax genotyping, for example, detecting multiple-clone infections and tracking the geographic origin of isolates.